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RNA-Binding Profiles of CKAP4 as an RNA-Binding Protein in Myocardial Tissues.

ABSTRACT: Background: Pathological tissue remodeling such as fibrosis is developed in various cardiac diseases. As one of cardiac activated-myofibroblast protein markers, CKAP4 may be involved in this process and the mechanisms have not been explored. Methods: We assumed that CKAP4 held a role in the regulation of cardiac fibrotic remodeling as an RNA-binding protein. Using improved RNA immunoprecipitation and sequencing (iRIP-seq), we sought to analyze the RNAs bound by CKAP4 in normal atrial muscle (IP1 group) and remodeling fibrotic atrial muscle (IP2 group) from patients with cardiac valvular disease. Quantitative PCR and Western blotting were applied to identify CKAP4 mRNA and protein expression levels in human right atrium samples. Results: iRIP-seq was successfully performed, CKAP4-bound RNAs were characterized. By statistically analyzing the distribution of binding peaks in various regions on the reference human genome, we found that the reads of IP samples were mainly distributed in the intergenic and intron regions implying that CKAP4 is more inclined to combine non-coding RNAs. There were 913 overlapping binding peaks between the IP1 and IP2 groups. The top five binding motifs were obtained by HOMER, in which GGGAU was the binding sequence that appeared simultaneously in both IP groups. Binding peak-related gene cluster enrichment analysis demonstrated these genes were mainly involved in biological processes such as signal transduction, protein phosphorylation, axonal guidance, and cell connection. The signal pathways ranking most varied in the IP2 group compared to the IP1 group were relating to mitotic cell cycle, protein ubiquitination and nerve growth factor receptors. More impressively, peak analysis revealed the lncRNA-binding features of CKAP4 in both IP groups. Furthermore, qPCR verified CKAP4 differentially bound lncRNAs including LINC00504, FLJ22447, RP11-326N17.2, and HELLPAR in remodeling myocardial tissues when compared with normal myocardial tissues. Finally, the expression of CKAP4 is down-regulated in human remodeling fibrotic atrium. Conclusions: We reveal certain RNA-binding features of CKAP4 suggesting a relevant role as an unconventional RNA-binding protein in cardiac remodeling process. Deeper structural and functional analysis will be helpful to enrich the regulatory network of cardiac remodeling and to identify potential therapeutic targets.

SUBMITTER: Zhu H

PROVIDER: S-EPMC8733325 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundMotivated by the increased amount of data on protein-RNA interactions and the availability of complete genome sequences of several organisms, many computational methods have been proposed to predict binding sites in protein-RNA interactions. However, most computational methods are limited to finding RNA-binding sites in proteins instead of protein-binding sites in RNAs. Predicting protein-binding sites in RNA is more challenging than predicting RNA-binding sites in proteins. Recent computational methods for finding protein-binding sites in RNAs have several drawbacks for practical use.ResultsWe developed a new support vector machine (SVM) model for predicting protein-binding regions in mRNA sequences. The model uses sequence profiles constructed from log-odds scores of mono- and di-nucleotides and nucleotide compositions. The model was evaluated by standard 10-fold cross validation, leave-one-protein-out (LOPO) cross validation and independent testing. Since actual mRNA sequences have more non-binding regions than protein-binding regions, we tested the model on several datasets with different ratios of protein-binding regions to non-binding regions. The best performance of the model was obtained in a balanced dataset of positive and negative instances. 10-fold cross validation with a balanced dataset achieved a sensitivity of 91.6%, a specificity of 92.4%, an accuracy of 92.0%, a positive predictive value (PPV) of 91.7%, a negative predictive value (NPV) of 92.3% and a Matthews correlation coefficient (MCC) of 0.840. LOPO cross validation showed a lower performance than the 10-fold cross validation, but the performance remains high (87.6% accuracy and 0.752 MCC). In testing the model on independent datasets, it achieved an accuracy of 82.2% and an MCC of 0.656. Testing of our model and other state-of-the-art methods on a same dataset showed that our model is better than the others.ConclusionsSequence profiles of log-odds scores of mono- and di-nucleotides were much more powerful features than nucleotide compositions in finding protein-binding regions in RNA sequences. But, a slight performance gain was obtained when using the sequence profiles along with nucleotide compositions. These are preliminary results of ongoing research, but demonstrate the potential of our approach as a powerful predictor of protein-binding regions in RNA. The program and supporting data are available at http://bclab.inha.ac.kr/RBPbinding .

Project description:BackgroundCircular RNA (circRNA) is one type of noncoding RNA that forms a covalently closed continuous loop. Similar to long noncoding RNA (lncRNA), circRNA can act as microRNA (miRNA) 'sponges' to regulate gene expression, and its abnormal expression is related to diseases such as atherosclerosis, nervous system disorders and cancer. So far, there have been no systematic studies on circRNA abundance and expression profiles in human adult and fetal tissues.ResultsWe explored circRNA expression profiles using RNA-seq data for six adult and fetal normal tissues (colon, heart, kidney, liver, lung, and stomach) and four gland normal tissues (adrenal gland, mammary gland, pancreas, and thyroid gland). A total of 8120, 25,933 and 14,433 circRNAs were detected by at least two supporting junction reads in adult, fetal and gland tissues, respectively. Among them, 3092, 14,241 and 6879 circRNAs were novel when compared to the published results. In each adult tissue type, we found at least 1000 circRNAs, among which 36.97-50.04% were tissue-specific. We reported 33 circRNAs that were ubiquitously expressed in all the adult tissues we examined. To further explore the potential "housekeeping" function of these circRNAs, we constructed a circRNA-miRNA-mRNA regulatory network containing 17 circRNAs, 22 miRNAs and 90 mRNAs. Furthermore, we found that both the abundance and the relative expression level of circRNAs were higher in fetal tissue than adult tissue. The number of circRNAs in gland tissues, especially in mammary gland (9665 circRNA candidates), was higher than that of other adult tissues (1160-3777).ConclusionsWe systematically investigated circRNA expression in a variety of human adult and fetal tissues. Our observation of different expression level of circRNAs in adult and fetal tissues suggested that circRNAs might play their role in a tissue-specific and development-specific fashion. Analysis of circRNA-miRNA-mRNA network provided potential targets of circRNAs. High expression level of circRNAs in mammary gland might be attributed to the rich innervation.

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RNA-Binding Profiles of CKAP4 as an RNA-Binding Protein in Myocardial Tissues.

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